Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates

ABSTRACT

The present invention relates to novel peptides that may be used in whole or in combination for the detection of  Mycobacterium tuberculosis  infection. In particular, the present invention relates to compositions and methods involving detection of antibodies contained in the blood of non-human primates that arise from an infection from  M. tuberculosis  or vaccination using an epitope specific inoculation. More particularly, the present invention provides a means to distinguish early, active, and latent  M. tuberculosis  infection. More particularly, the present invention describes an immunological diagnostic mechanism for the detection of  M. tuberculosis  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage of International (PCT) PatentApplication Serial No. PCT/US2013/025072, filed Feb. 7, 2013, whichclaims the benefit of and priority to U.S. Provisional PatentApplication No. 61/595,805, filed Feb. 7, 2012, and to U.S. ProvisionalPatent Application No. 61/723,094, filed Nov. 6, 2012, the contents ofeach of which are hereby incorporated by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel peptides that may be used inwhole or in combination for the detection of Mycobacterium tuberculosisinfection. In particular, the present invention relates to compositionsand methods involving detection of antibodies contained in the blood ofnon-human primates that arise from an infection from M. tuberculosis orvaccination using an epitope specific inoculation.

BACKGROUND OF THE INVENTION

The intracellular bacillus pathogen Mycobacterium tuberculosis (M. tb)causes the fatal and rapidly spreading disease tuberculosis (TB) innon-human primates (NHP), similar to active TB in humans. In NHPs, M. tbis typically spread by inhalation of aerosolized infectious particlesexpelled by infected individuals (humans and animals) [1]. Because ofthe health threat to captive primates and the zoonotic potential to andfrom their human caretakers, strict screening protocols have beenimplemented to prevent outbreaks[2]. Despite the precautions taken toprevent M. tb exposure in captive primate colonies, outbreaks continueto cause a major impact on colony health. Outbreaks result in animallosses, disruption of research, potential exposure to human caretakers,and the immeasurable costs to contain the outbreaks [3-11].

Current methods for TB screening in non-human primates rely on thebi-annual intradermal tuberculin skin test (TST), a test that has beenroutinely used for decades [2, 12]. Injection of M. tb antigen istypically performed in the eyelid, and relies on immune cell mediatedrecognition of the antigens generated by previous exposure to M. tb. Theantigens currently used in the TST are semi-purified heterogeneous crudeprotein preparations of either mammalian old tuberculin (MOT) orpurified protein derivative (PPD). The cell mediated response resultingin local inflammation and necrotic tissue measured by a TST takes atleast 4 weeks after infection to develop and has some severe limitationsin terms of diagnostic sensitivity and specificity. Additionally, theTST is labor intensive, and requires increased animal handling over thethree days it takes to develop the response of a swelled eyelid ornecrotic tissue.

While false positive TST results in loss of individually valuableresearch animals, the greatest threat to NHP colony and human health arefalse negative results, which make entire colonies vulnerable tooutbreaks [5, 10, 13]. Because NHPs can develop latent TB withoutobvious clinical symptoms [3, 7, 14-15], false negative results oranergy to TST pose a significant threat to colony health when combinedwith the low diagnostic sensitivity of the TST. Anergy can also beinduced in research primates by immunosuppressive treatment or disease,or concurrent viral infection like measles [4, 9, 16-17]. Additionally,reports of anergy to TST following measles vaccination complicates usingthe TST as part of importation quarantine procedure[18]. Despite the TSTbeing USDA-approved for diagnostic testing of NHPs for TB, it is widelyconsidered that a negative TST result does not reliably excludetuberculosis infection (reports between 79 and 90% predictivevalue)[19]. It is, however, the standard for antemortem diagnostictesting and requires lengthy postmortem confirmation by the “goldstandard” of mycobacterial culture and isolation.

Other immune-based screening assays and serological tests, includinginterferon gamma release assays (IGRA) and commercial antibody detectionassays can have a higher sensitivity than the TST for acute infection,but rely on similar sets of M. tb recombinant proteins that often failto identify latently infected animals. In the case of the commerciallyavailable IGRA test PRIMAGAM (Prionics AG, Zurich, Switzerland), thesemi-purified PPD antigen set is used to stimulate IFN-γ release bywhole blood leukocytes[19]. The PrimaTB STAT-PAK assay is a lateral flowdevice for measuring presence of TB-specific antibodies in NHP plasma orserum, and uses an antigen cocktail of recombinant antigens includingESAT-6, CFP-10, MPB83, and TBF10 (a fusion protein from CorixaCorp)[20]. Relative to the TST these tests are expensive, technicallydifficult, and impractical to perform on a colony-wide basis[19].Moreover, these methods all rely on semi-purified or recombinantantigens for identifying a biological or antibody response, all aresubject to the same limitations of sensitivity. At best, reportedpredictive value in experimentally infected animals is between 83 and94% for PRIMAGAM and 80 and 94% for PrimaTB STAT-PAK, respectively[19-21]. Unpublished studies have reported significantly lowerpredictive value (<50%) for naturally occurring infections in NHPs(Table 2). Current recommendations for TB testing include routine TST inconjunction with IGRA or antibody assays for routine screening becauseno one test is both sensitive and specific enough[22].

What is needed in the art are improved devices and methods fordetermining if a subject animal has tuberculosis, and especially devicesand methods which allow for accurate, specific and sensitive monitoringof primate colonies.

SUMMARY OF THE INVENTION

The present invention relates to novel peptides that may be used inwhole or in combination for the detection of Mycobacterium tuberculosisinfection. In particular, the present invention relates to compositionsand methods involving detection of antibodies contained in the blood ofnon-human primates that arise from an infection from M. tuberculosis orvaccination using an epitope specific inoculation. More particularly,the present invention provides a means to distinguish early, active, andlatent M. tuberculosis infection. More particularly, the presentinvention describes an immunological diagnostic mechanism for thedetection of M. tuberculosis infection.

Accordingly, in some embodiments, the present invention provides animmunoassay device comprising one or more capture reagents comprising apeptide selected from the group consisting of SEQ ID NOs.1-149. In someembodiments, the device comprises two or more capture reagents, eachcomprising a different peptide selected from the group consisting of SEQID NOs.1-149. In some embodiments, the device comprises three or morecapture reagents, each comprising a different peptide selected from thegroup consisting of SEQ ID NOs.1-149. In some embodiments, the devicecomprises four or more capture reagents, each comprising a differentpeptide selected from the group consisting of SEQ ID NOs.1-149. In someembodiments, the device comprises five or more capture reagents, eachcomprising a different peptide selected from the group consisting of SEQID NOs.1-149. In some embodiments, the device comprises ten or morecapture reagents, each comprising a different peptide selected from thegroup consisting of SEQ ID NOs.1-149. In some embodiments, the devicecomprises one or more capture reagents comprising isolated peptidesselected from SEQ ID NOs: 28, 98, 141, 145 and combinations thereof. Insome embodiments, the device comprises two or more distinct capturereagents, each comprising a different peptide selected from the groupconsisting of SEQ ID NOs.141, 145, 147, 98, 139 and 56. In someembodiments, the device comprises three or more distinct capturereagents, each comprising a different peptide selected from the groupconsisting of SEQ ID NOs.141, 145, 147, 98, 139 and 56. In someembodiments, the device comprises four or more distinct capturereagents, each comprising a different peptide selected from the groupconsisting of SEQ ID NOs.141, 145, 147, 98, 139 and 56. In someembodiments, the device comprises a first capture agent comprising SEQID NO:141, a second capture reagent comprising SEQ ID NO:145, a thirdcapture reagent comprising SEQ ID NO: 147; and a fourth capture reagentcomprising SEQ ID NO:98.

In some embodiments, the capture reagent is selected from the groupconsisting of proteins and protein conjugates. In some embodiments, theone or more capture reagents comprise a polypeptide R₁—X—R₂, wherein Xis a peptide selected from the group consisting of SEQ ID NOs.1-149, R₁is selected from the group consisting of the amino terminus of saidpolypeptide, an amino acid or a polypeptide chain, and R₂ is selectedfrom the group consisting of the amino terminus of said polypeptide, anamino acid or a polypeptide chain. In some embodiments, R₁ is apolypeptide chain of from about 2 to about 100 amino acids in length. Insome embodiments, R₁ is a polypeptide chain of from about 2 to about 20amino acids in length. In some embodiments, R₂ is a polypeptide chain offrom about 2 to about 100 amino acids in length. In some embodiments, R₂is a polypeptide chain of from about 2 to about 20 amino acids inlength.

In some embodiments, the devices further comprise a surface. In someembodiments, the at least one capture reagent is displayed on saidsurface. In some embodiments, the devices further comprise at least twoof said capture reagents arrayed on said surface.

In some embodiments, the immunoassay device is selected from the groupconsisting of a chip-based array, an ELISA device, a lateral flow deviceand beads.

In some embodiments, the present invention provides methods ofmonitoring, detecting or diagnosing infection by Mycobacteriumtuberculosis in a subject or subjects comprising: contacting animmunoassay device as described above with a biological sample from saidsubject, and detecting the presence of antibodies in said biologicalsample that bind to said at least one capture reagent. In someembodiments, the subject is a human. In some embodiments, the subject isa non-human primate. In some embodiments, the sample is a blood or serumsample. In some embodiments, the methods further comprise analyzingmultiple biological samples from a non-human primate colony. In someembodiments, the methods further comprise contacting said antibodies insaid biological sample that bind to said at least one capture reagentthat is detectably labeled for direct or indirect detection. In someembodiments, the detection reagent is labeled with a detectablecomponent selected from the group consisting of a fluorescent compound,a luminescent compound, an enzyme, a radiolabel, and a hapten. In someembodiments, the detection reagent is an antigen binding protein.

In some embodiments, the present invention provides methods ofmonitoring, detecting or diagnosing infection by Mycobacteriumtuberculosis in a subject or subjects comprising: detecting the presenceof antibodies in a biological sample from said subject or subjects thatbind to an antigenic composition comprising a peptide selected from thegroup consisting of SEQ ID NOs.1-149. In some embodiments, the methodscomprise detecting the presence of two or more antibodies in saidbiological sample that bind two or more antigenic compositions, eachcomprising a different peptide selected from the group consisting of SEQID NOs.1-149. In some embodiments, the methods comprise detecting thepresence of three or more antibodies in said biological sample that bindthree or more antigenic compositions, each comprising a differentpeptide selected from the group consisting of SEQ ID NOs.1-149. In someembodiments, the methods comprise detecting the presence of four or moreantibodies in said biological sample that bind four or more antigeniccompositions, each comprising a different peptide selected from thegroup consisting of SEQ ID NOs.1-149. In some embodiments, the methodscomprise detecting the presence of five or more antibodies in saidbiological sample that bind five or more antigenic compositions, eachcomprising a different peptide selected from the group consisting of SEQID NOs.1-149. In some embodiments, the methods comprise detecting thepresence of ten or more antibodies in said biological sample that bindten or more antigenic compositions, each comprising a different peptideselected from the group consisting of SEQ ID NOs.1-149.

In some embodiments, antigenic composition is selected from the groupconsisting of proteins and protein conjugates. In some embodiments, theone or more antigenic compositions comprise a polypeptide R₁—X—R₂,wherein X is a peptide selected from the group consisting of SEQ IDNOs.1-149, R₁ is selected from the group consisting of the aminoterminus of said polypeptide, an amino acid or a polypeptide chain, andR₂ is selected from the group consisting of the amino terminus of saidpolypeptide, an amino acid or a polypeptide chain. In some embodiments,R₁ is a polypeptide chain of from about 2 to about 100 amino acids inlength. In some embodiments, R₁ is a polypeptide chain of from about 2to about 20 amino acids in length. In some embodiments, R₂ is apolypeptide chain of from about 2 to about 100 amino acids in length. Insome embodiments, R₂ is a polypeptide chain of from about 2 to about 20amino acids in length. In some embodiments, the methods further comprisedetecting the presence of antibodies in said sample that bind to atleast two different antigenic compositions each comprising a differentpeptide selected from the group consisting of SEQ ID NOs:141, 145, 147,98, 139 and 56. In some embodiments, the methods further comprisedetecting the presence of antibodies in said sample that bind to atleast three different antigenic compositions each comprising a differentpeptide selected from the group consisting of SEQ ID NOs: 141, 145, 147,98, 139 and 56. In some embodiments, the methods further comprisedetecting the presence of antibodies in said sample that bind to atleast four different antigenic compositions each comprising a differentpeptide selected from the group consisting of SEQ ID NOs: 141, 145, 147,98, 139 and 56. In some embodiments, the methods further comprisedetecting the presence of antibodies in said sample that bind to a firstantigenic composition comprising SEQ ID NO:141, a second antigeniccomposition comprising SEQ ID NO:145, a third antigenic compositioncomprising SEQ ID NO: 147; and a fourth antigenic composition comprisingSEQ ID NO:98. In some embodiments, the methods further comprisedetecting the presence of antibodies in said sample that bind to a fifthantigenic composition comprising SEQ ID NO:139 and a sixth antigenicagent comprising SEQ ID NO:56.

In some embodiments, the at least one antigenic composition is displayedon a device comprising a surface. In some embodiments, at least two ofsaid antigenic compositions are arrayed on said surface. In someembodiments, the surface is a component of a device selected from thegroup consisting of a chip-based array, an ELISA device, a lateral flowdevice and beads.

In some embodiments, the method has a specificity of greater than about80%. In some embodiments, the method has a specificity of greater thanabout 90%. In some embodiments, the method has a specificity of greaterthan about 95%. In some embodiments, the method has a sensitivity ofgreater than about 80%. In some embodiments, the method has asensitivity of greater than about 90%. In some embodiments, the methodhas a sensitivity of greater than about 95%.

In some embodiments, the present invention provides a panel of antigeniccompositions comprising two or more antigenic compositions, eachcomprising a different peptide selected from the group consisting of SEQID NOs.1-149.

In some embodiments, the present invention provides an immunogeniccomposition comprising one more peptides selected from the groupconsisting of peptides identified by SEQ ID NOs:1-149. In someembodiments, the immunogenic compositions further comprise an adjuvant.In some embodiments, the immunogenic compositions comprise a fusionpolypeptide comprising two or more peptides identified by SEQ IDNOs:1-149.

In some embodiments, the present invention provides methods ofvaccinating a subject comprising administering the immunogeniccomposition at described above to a subject.

In some embodiments, the present invention provides for the use of theassay devices described above to detect, monitor or diagnose infectionor exposure to Mycobacterium tuberculosis in a subject or subjects,particularly a non-human primate or a colony of non-human primates.

In some embodiments, the present invention provides for the use of theimmunogenic compositions described above to detect, monitor or diagnoseinfection or exposure to Mycobacterium tuberculosis in a subject orsubjects, particularly a non-human primate or a colony of non-humanprimates.

In some embodiments, the present invention provides for the use ofimmunogenic compositions described above to vaccinate a subject orsubjects, particularly a non-human primate or a colony of non-humanprimates.

In some embodiments, the one or more of the peptides or conjugatesdescribed above (alone or in combination) are used as an antigenstimulation mixture for cell based assays including, but not limited to,cytokine release assays (particularly interferon gamma release andinterleukin 12) as measured by ELISA, Elispot, or bead based methods. Inother embodiments, the peptides or conjugates described above (alone orin combination) are used in T-cell capture assays. In still otherembodiments, the peptides or conjugates described above (alone or incombination) are used as an antigenic substitute for tuberculin in thetuberculin skin test (TST).

DESCRIPTION OF THE FIGURES

FIG. 1 provides an analysis of positive and negative serum samples fromTulane National Primate Research Center (time course infection study)using a multiplexed Ag microarray with individual and mixed peptide-BSAconjugates. A: Peptide 4; B: Peptide 6; C: Peptide 15; D: Peptide 20; E:Mix A.

FIG. 2 provides an ROC analysis of 141 negative and 35 positive serumsamples.

FIG. 3. The Relative Intensity Unit (RIU) is reported for the Mix ofpeptides (Peptides 6, 15, 17, and 20) and for each peptide individually.Values highlighted in grey show higher signal than the Naïve pooledsample.

DEFINITIONS

The terms “detecting” or “detection” or “determining the level” refer toquantitatively or non-quantitatively determination of the presence ofthe analyte(s) under investigation (e.g., AMH). “Detecting Formation ofa Complex” refers to detecting a complex comprising a detector reagentby any method suitable for observing the particular label associatedwith the detector reagent; for instance, visual observation of a colored(or otherwise visible) label, measurement or visual detection of afluorescent, chemiluminescent or radioactive label.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include urine andblood products, such as plasma, serum and the like. Such examples arenot however to be construed as limiting the sample types applicable tothe present invention.

As used herein, the terms “protein,” “polypeptide,” and “peptide” referto a molecule comprising amino acids joined via peptide bonds. Ingeneral “peptide” is used to refer to a sequence of 20 or less aminoacids and “polypeptide” is used to refer to a sequence of greater than20 amino acids. The proteins, polypeptides and peptides may be natural,produced by a recombinant process (i.e., expression of exogenous nucleicacid encoding the peptide, polypeptide or protein in an organism, hostcell, or cell-free system) or produced by chemical synthesis.

The term “carrier” refers to a molecule that allows conjugation ofadditional, usually smaller, molecules such as peptides.

The term “specific binding partner (or binding partner)” refers to amember of a pair of molecules that interact by means of specific,noncovalent interactions that depend on the three-dimensional structuresof the molecules involved. Typical pairs of specific binding partnersinclude antigen/antibody, hapten/antibody, hormone/receptor, nucleicacid strand/complementary nucleic acid strand, substrate/enzyme,inhibitor/enzyme, carbohydrate/lectin, biotin/(strept)avidin, andvirus/cellular receptor.

As used herein, the terms “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)2 fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg) Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

The term “label” refers to a molecule or composition bound to ananalyte, analyte analog, detector reagent, or binding partner that isdetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Examples of labels, includingenzymes, colloidal gold particles, colored latex particles, have beendisclosed (U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and4,954,452, each incorporated by reference herein). Additional examplesof useful labels include, without limitation, radioactive isotopes,co-factors, ligands, chemiluminescent or fluorescent agents,protein-adsorbed silver particles, protein-adsorbed iron particles,protein-adsorbed copper particles, protein-adsorbed selenium particles,protein-adsorbed sulfur particles, protein-adsorbed tellurium particles,protein-adsorbed carbon particles, and protein-coupled dye sacs. Theattachment of a compound (e.g., a detector reagent) to a label can bethrough covalent bonds, adsorption processes, hydrophobic and/orelectrostatic bonds, as in chelates and the like, or combinations ofthese bonds and interactions and/or may involve a linking group.

The phrase “label-free detection” refers to the detection of animmunoglobulin or antibody without the addition of a label to theimmunoglobulin or a primary or secondary affinity binder that contains alabel. Examples of label-free detection may be Surface Plasmon Resonance(SPR), Quartz Crystal Microbalance (QCM), and Optical Interferometry.

The phrase “specifically binds to an analyte” or “specificallyimmunoreactive with,” when referring to an antibody or polypeptide,refers to a binding reaction which is determinative of the presence ofthe analyte (which can be an antibody or polypeptide) in the presence ofa heterogeneous population of molecules such as proteins and otherbiologic molecules. Thus, under designated immunoassay conditions, thespecified antibodies or polypeptides bind to a particular analyte and donot bind in a significant amount to other analytes present in thesample. A variety of immunoassay formats may be used to selectantibodies or polypeptides specifically immunoreactive with a particularanalyte. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies polypeptides specifically immunoreactivewith a protein or antibody See Harlow and Lane, Antibodies, A LaboratoryManual, CSHP, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

The term “capture agent” refers to an unlabeled specific binding partnerthat is specific for (i) an analyte, such as an immunoglobulin that hasbeen produced by an animal in response to infection by Mycobacteriumtuberculosis, or (ii) a detector reagent or an analyte, as in acompetitive assay, or for (iii) an ancillary specific binding partner,which itself is specific for the analyte, as in an indirect assay. Asused herein, an “ancillary specific binding partner” is a specificbinding partner that binds to the specific binding partner of ananalyte. For example, an ancillary specific binding partner may includean antibody specific for another antibody, for example, goat anti-humanantibody. A “capture area” is a region of a lateral flow device wherethe capture reagent is immobilized. A lateral flow device may have morethan one capture area, for example, a “primary capture area,” a“secondary capture area,” and so on. Often a different capture reagentwill be immobilized in the primary, secondary, or other capture areas.Multiple capture areas may have any orientation with respect to eachother on the lateral flow substrate; for example, a primary capture areamay be distal or proximal to a secondary (or other) capture area andvice versa. Alternatively, a primary capture area and a secondary (orother) capture area may be oriented perpendicularly to each other suchthat the two (or more) capture areas form a cross or a plus sign orother symbol.

The term “detector reagent” refers to a specific binding partner that isconjugated to a label. Detector reagents include, for example, labeledanalyte-specific binding members or labeled ancillary specific bindingmembers (such as enzyme-conjugate, goat anti-primate antibodies).

The term “lateral flow device” refers to an analytical device in theform of a test strip used in lateral flow chromatography, in which atest sample fluid, suspected of containing an analyte, flows (forexample by capillary action) through the strip (which is frequently madeof bibulous materials such as paper, nitrocellulose, and cellulose). Thetest fluid and any suspended analyte can flow along the strip to adetection zone in which the analyte (if present) interacts with adetection agent to indicate a presence, absence and/or quantity of theanalyte.

The term “sample application area” refers to an area where a fluidsample is introduced to an immunochromatographic test strip, such as animmunochromatographic test strip present in a lateral flow device. Inone example, the sample may be introduced to the sample application areaby external application, as with a dropper or other applicator. Inanother example, the sample application area may be directly immersed inthe sample, such as when a test strip is dipped into a container holdinga sample. In yet another example, the sample may be poured or expressedonto the sample application area.

The term “solid support” or “substrate” means material which isinsoluble, or can be made insoluble by a subsequent reaction. Numerousand varied solid supports are known to those in the art and include,without limitation, nitrocellulose, the walls of wells of a reactiontray, multi-well plates, test tubes, polystyrene beads, magnetic beads,membranes, microparticles (such as latex particles), and sheep (or otheranimal) red blood cells. Any suitable porous material with sufficientporosity to allow access by detector reagents and a suitable surfaceaffinity to immobilize capture reagents is contemplated by this term.For example, the porous structure of nitrocellulose has excellentabsorption and adsorption qualities for a wide variety of reagents, forinstance, capture reagents. Nylon possesses similar characteristics andis also suitable. Microporous structures are useful, as are materialswith gel structure in the hydrated state.

Further examples of useful solid supports include: natural polymericcarbohydrates and their synthetically modified, cross-linked orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers which may be prepared with suitably porousstructures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

It is contemplated that porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

The surface of a solid support may be activated by chemical processesthat cause covalent linkage of an agent (e.g., a capture reagent) to thesupport. However, any other suitable method may be used for immobilizingan agent (e.g., a capture reagent) to a solid support including, withoutlimitation, ionic interactions, hydrophobic interactions, covalentinteractions and the like. The particular forces that result inimmobilization of an agent on a solid phase are not important for themethods and devices described herein.

Except as otherwise physically constrained, a solid support may be usedin any suitable shapes, such as films, sheets, strips, or plates, or itmay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

A “lateral flow substrate” is any solid support or substrate that isuseful in a lateral flow device.

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

As used herein, the terms “protein microarray” and “protein chip” referto protein-detecting molecules immobilized at high density on asubstrate, and probed for various biochemical activities. (See, forexample: Zhu H and Snyder M, “Protein chip technology”, Current Opinionin Chemical Biology 7: 55-63, 2003; Cutler P, “Protein arrays: Thecurrent state of the art”, Proteomics 3; 3-18, 2003; and MacBeath G,“Protein microarrays and proteomics”, Nature Genetics Supplement 32:526-532, 2002, each of which is incorporated herein by reference in itsentirety).

As used herein, the term “sensitivity” when used in reference to anassay refers to the proportion of actual positives which are correctlyidentified as such (e.g., the percentage of infected, latent orsymptomatic subjects who are correctly identified as having thecondition). Sensitivity may be calculated as the number of truepositives divided by the sum of the number of true positives and thenumber of false negatives.

As used herein the term “specificity” when used in relation to an assayrefers to proportion of negatives which are correctly identified (e.g.,the percentage of healthy subjects who are correctly identified as nothaving the condition). Specificity may be calculated as the number oftrue negatives divided by the sum of the number of true negatives andthe number of false positives.

“Blocking agent” means a molecular arrangement that will absorb to thesurface of the substrate. The absorption can result because ofnon-covalent bonding attractive forces or because the blocking agentcontains a reactive group. For example, a polyethylene glycol group canact as a blocking agent when it is covalently bonded to the surface orthe protein bovine serum albumin can act as a blocking agent when it isnon-covalently attached to the surface.

“Fusion protein” means a protein that contains additional moleculararrangements including, but not limited to, naturally or non-naturallyamino acids usually the result of producing the protein by manipulatingbiological processes.

“Linker” means a molecular arrangement with a reactive group that bindsa biological entity by exposure to the reactive group resulting in abiological entity linked to the molecular arrangement. The linkerincludes the molecular arrangements before and after the reactive groupbinds to the biological entity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel peptides that may be used inwhole or in combination for the detection of Mycobacterium tuberculosisand related infections, including Mycobacterium bovis. In particular,the present invention relates to compositions and methods involvingdetection of antibodies contained in the blood of human or non-humanprimates that arise from an infection from M. tuberculosis orvaccination using an epitope specific inoculation. More particularly,the present invention provides a means to distinguish early, active, andlatent M. tuberculosis infection. More particularly, the presentinvention describes an immunological diagnostic mechanism for thedetection of M. tuberculosis infection.

Historically, antigens used for TB immunoassays in non-human primateshave focused on secreted antigens that have been shown to havediagnostic potential in humans. Work by Brusasca et al. first used 14proteins identified as immunodominant in human TB to identify theantibody response to these proteins in experimentally infected NHPs[23].Of these 14, only 3 proteins (ESAT-6, α-crystallin, and CFP-10)identified antibodies in more than 85% of infected animals with anantibody reaction. Importantly, the most seroreactive human antigen atthe time of that study (38 kDa, Rv0934) was only reactive with 14% ofinfected animals, highlighting the need for NHP specific antigendiscovery. More recent studies have shown that individual recombinantproteins previous used in commercial immunochromatographic kits for bothhuman and NHPs have at best 75% diagnostic sensitivity, and several TSTpositive animals were seronegative to the panel of 10 recombinantproteins [24]. Our preliminary studies showed that neither purifiedrecombinant proteins (ESAT-6 and 16 kDa α-crystallin) nor theheterogeneous TST antigen MOT were able to accurately distinguishbetween positive and negative samples in our microarray based assay forantibody detection. Overall, current TB tests are limited by the poorlydefined antigens for NHP testing and cannot use those antigenssuccessfully in humans.

Accordingly, the present invention provides individual peptides,polypeptides, and proteins as well as panels of peptides, polypeptides,and proteins which are useful detecting, monitoring or diagnosinginfection by Mycobacterium tuberculosis and for making vaccines toprotect against infection by Mycobacterium tuberculosis. In someembodiments, the peptides, proteins, or polypeptides are encoded by orcomprise SEQ ID Nos.: 1-149 or are at least 80%, 90%, 95% or 99%identical to SEQ ID NOs.:1-149 and have the property of binding to anantibody that recognizes the corresponding unaltered sequence.

Sequence ID Peptide Sequence 1 AAATHSPRNVQFYCI 2 AAIPLLTAPVIGMAM 3AAQVWSQCHSYGFDA 4 ADEEQQQALSSQMGF 5 AFEWYYQSGLSIVMP 6 AIGLGGGAGGDGGQG 7AKLIIWYPHYAWLLL 8 AMEDLVRAYHAMSST 9 ANNWYQPARRGFSTG 10 APAPIASPIPVGAPG11 AREVVELDRDEAMRL 12 ASQSTTNPIFGMPSP 13 ASVSIDGAQQASVAL 14DAERFGARLVASPQH 15 DALAGKGASAIVLRG 16 DGITLFNPAAVPIRR 17 DGTHQTLQGADLTVIPeptide 2 18 DQDTAELHRRATHFR 19 DVSVAQGVTNLHTAI 20 DYAIVGGAPGSAPRE 21EARRADLGCDIVDAT 22 EFPDLEGQVQDLAAQ 23 EGLWAGVVIPESGRA 24 ESPAPHVPSHGPHQP25 FDETIDKSTDKTLHS 26 FDWRYPPSPPPQPTQ 27 FLIIDGWPGFVGEFP 28FPQYRELGVYLVRGI Peptide 4 29 FQIHDPTTKDRQGND 30 FSIVRRSSRPEPSPR 31GAGGSLTRTPLMSQL 32 GAGSGFANFGSLGSG 33 GCGTCNIAEALKMSL 34 GDFYAGEKSMTLDRA35 GFDFTAQGVWAFARP 36 GGAPQTGKSTLLQTM 37 GGARFAVEQGHGVAL 38GGREGPTAQISAGFC 39 GIAFGLFIVTNAAKL 40 GLDPVNYQNFAVTND 41 GLYNTGGLPPGTPAV42 GNAGLFGDGGAGGDG 43 GPSIQARLRAEEASG 44 GRGAPEDAPLIMRGK 45GTTQFGDTTANKDAW 46 HLAKGELRSIHNRTD 47 HLREKGVKLEAQRPH 48 HRHPSANQQMLWTLV49 HVKPTGSAVVGLSMA 50 HYLHVRPADQFDAMI 51 IELTKRTLWSGLDAA 52ILLECTMAGMATCTL 53 IPIGLRETDLTPAHC 54 ITYQAWQAQWNQAME 55 IVGAAGGMPPMAPLA56 KGRSSEMIICGGVNI Peptide 9 57 KVKPQKPKATKPPKV 58 KYVEADLRVLDEIAP 59LAAPVDPSTPAPSTT 60 LAIPGTNWIGQAAEA 61 LDWERNFLRQLQSHR 62 LGGGVAANLGRAASV63 LGNSVYTSNAQLVVY Peptide 10 64 LGQPRSYLAPPTRPA 65 LLSQGKFPYKSSWIE 66LNTARLMAGAGPAPM 67 LPGTAVANPVDPARI 68 LQVIERTWRYLKVPC 69 LRCGDFALGGPQGRG70 LRILKTDIYAPTGAV Peptide 11 71 LTYWTAGDTRNRGRE 72 MAGDTTITIVGNLTA 73MASGSGLCKTTSNFI 74 MELLDAFGIAMAGAP 75 MGTTLTAILFAGNRL 76 MIDGVYKVCKGLEKI77 MKLTTMIKTAVAVVA Peptide 12 78 MNSAIIKIAKWAQSQ 79 MQLVDRVRGAVTGMS 80MRLSLTALSAGVGAV 81 MSAPAVAAGPTAAGA 82 NAEIAATIANGGITM 83 NARTADGINYRVLWQ84 NDPDRASMLFEGSTI 85 NKPVISWAGDNGIYF 86 NVDGAQREIDILEND 87NVDGAQREIDILEND 88 PANRPGRAVSMEKHH 89 PLDDGDVIDSMFMSK 90 PLQALQTVQQNVLTV91 PPAATQTLGQLGEMS 92 PPGDERHMLWFELMK 93 PPIRAPGGDAADTRL 94PPTPPMPIAGPEPAP 95 PSGGPVAASAAPSIP 96 PSGTAVGAGARSSVG 97 QAQLISSQAQQGGQQ98 QEAGNFERISGDLKT Peptide 20 99 QMQDAFETGVMFSLH 100 QSGPAHADESAASVT 101RGIPPGHVGVAWRGT 102 RNIIDMHLPRHRLDS 103 RQRRTKGAGGSFTSR 104RRHLQDVWGVDVSGA Peptide 14 105 RSEPDKVNRVVAEMQ 106 RTYSQIDDGAAGVFA 107SAASVTPAAASGVPG 108 SADDGTPVSMIPVSA 109 SAKMLSVVPLMAGGG 110SDVFTTITPATAQGI 111 SKAATDMLAYQYHKS 112 SKIPRGEEAGKLWDA 113SKLMTRIAGAGAMGS 114 SLFPEFSELFAAFPS 115 SMEKHHLMIGVPRFD 116SPGQQPGGGVPAQAM 117 SQRGWRHWVHALTRI 118 SSTHEANTMAMMARD 119STVSGVVVVASVSID 120 TASDFWGGAGSAACQ 121 TDAKLLSSILTYHVI 122TGQGNSLKVGNADVV 123 TLLQTMVMSAAATHS 124 TMLVVPVGAGPGLRE 125TNAAKLTVAVARIAL 126 TPKTKIETALDRQKI 127 TQAVLTATNFFGINT 128TRDKFLSAATSSTPR 129 TRLMRLEDEMKEGRY 130 TRQLHQLGLESSERE 131TTGPKPLVQDEYAKH 132 TYSQIDDGAAGVFAE 133 VHLARDLRLHRDVAV 134VLDESPEFDRTALNR 135 VLESKTVGDSCVVLE 136 VTSLFSQVGGTGGGN 137VVVGGSIDAAIDTAK 138 APHESFDTQYTRHVE Peptide 1 139 EAEHQAIISDVLTASPeptide 3 140 HARDVLADRRLFVLT Peptide 5 141 IHSLLDEGKQSLTKL Peptide 6AAAWGGSGSEAY 142 KELHLRLGTATADLS Peptide 7 143 KFRQLGDIVLELAAA Peptide 8144 NSLVTATHGANVSLV Peptide 13 145 TKLAAAWGGSGSEAY Peptide 15QGVQQKWDATAT 146 VWRRDLRAIVRLLAW Peptide 16 147 APRADLATREEQIAVPeptide 17 148 HDIQVTLSAGQSVTL Peptide 18 149 IRTNQVSTILASDGS Peptide 19

SEQ ID NOs.: 1-149 encode peptide sequences that serve as antigens whena subject is infected by Mycobacterium tuberculosis or relatedorganisms. The present invention encompasses these peptides in isolationas well as proteins, polypeptides, fusion proteins and proteinconjugates that comprise the identified peptide sequences. In preferredembodiments, the proteins, polypeptides, fusion proteins and proteinconjugates that comprise one or more of SEQ ID NOs:1-149 have theproperty of binding to antibodies that specifically recognize peptidesencoded by SEQ ID NOs:1-149. In some embodiments, polypeptidescomprising a peptide (or peptides) encoded by SEQ ID NOs: 1-149 comprisethe base peptide and from 1, 2, 5, 10, 20, 30, 40, or 50 to about 100 or200 amino acids on either or both of the amino- or carboxy-terminus ofthe base peptide. In some embodiments, polypeptides comprising a peptide(or peptides) encoded by SEQ ID NOs: 1-149 comprise the base peptide and1, 2, 5, at least 5, at least 10, at least 20, at least 30, at least 40,or at least 50 amino acids on either or both of the amino- orcarboxy-terminus of the base peptide.

In some embodiments, the present invention provides devices, kits andmethods for determining whether a subject has been exposed toMycobacterium tuberculosis or related organisms, infected byMycobacterium tuberculosis or related organisms or has tuberculosis orother disease. In some embodiments, the subject is a human or non-humanprimate. In some embodiments, the methods, kits and devices are used toassay the presence or amount of antibodies in the serum, blood or tissueof a subject to specified Mycobacterium tuberculosis peptides. A numberof methods and devices can be used to assay samples from subjects forthe presence of antibodies to Mycobacterium tuberculosis (Mtb),including, but not limited to, protein detection chips, bead-basedassays, lateral flow devices, and enzyme-linked immunosorbent assays(ELISAs). The assays may be singleplex assays or multiplex assays.

In singleplex assays, an antigenic composition or capture reagentcomprising one of the peptides encoded by SEQ ID NOs:1-149 is utilizedin the assay. In some embodiments, the capture reagent comprises anisolated peptide encoded by one of by SEQ ID NOs:1-149. For example, thecapture reagent may comprise the isolated peptide in addition to aconjugate or linker molecule. In some embodiments, the isolated basepeptide may be flanked by from 1, 2, 5, 10, 20, 30, 40, or 50 to about100 or 200 amino acids on either or both of the amino- orcarboxy-terminus of the base peptide. In multiplex assays, a panel ofantigenic compositions or capture reagents are utilized in the assay. Insome embodiments, the panel comprises at least 2, 3, 5, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80,90 or 100 or more of SEQ ID NOs: 1-149. In some embodiments, the panelcomprises 2, 3, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 of SEQ ID NOs: 1-149. Insome embodiments, the specific capture reagents utilized in themultiplex assay comprise an isolated peptide encoded by one of by SEQ IDNOs:1-149. For example, the capture reagent may comprise the isolatedpeptide in addition to a conjugate or linker molecule. In someembodiments, the isolated base peptide may be flanked by from 1, 2, 5,10, 20, 30, 40, or 50 to about 100 or 200 amino acids on either or bothof the amino- or carboxy-terminus of the base peptide. In someembodiments, the assays have a specificity of at least 80%, 90%, 95% or99%. In some embodiments, the assays have a sensitivity of at least 80%,90%, 95% or 99%. In some embodiments, the present invention provides anantigenic composition or capture reagent comprising one of the peptidesencoded by SEQ ID NOs:1-149 that is conjugated to a carrier molecule.

In some embodiments, the Mtb capture reagent of antigenic composition isbrought in contact with, and allowed to bind to, a solid support orcarrier, such as nitrocellulose or polystyrene or any other solidsupport known in the art (see below), allowing the antigens to adsorband become immobilized to the solid support. This immobilized antigen isthen allowed to interact with the biological fluid sample which is beingtested for the presence of anti-Mtb antibodies, such that any antibodiesin the sample will bind to the immobilized antigen. The support to whichthe antibody is now bound may then be washed with suitable buffers afterwhich a detectably labeled binding partner for the antibody isintroduced. The binding partner binds to the immobilized antibody.Detection of the label is a measure of the immobilized antibody. In someembodiments, the immunoassay of this invention may be a “two-site” or“sandwich” assay. The fluid containing the antibody being assayed isallowed to contact a solid support. After addition of the Mtbantigen(s), a quantity of detectably labeled soluble antibody is addedto permit detection and/or quantitation of the ternary complex formedbetween solid-phase antibody, antigen, and labeled antibody. Sandwichassays are described by Wide, Radioimmune Assay Method, Kirkham et al,Eds., E. & S. Livingstone, Edinburgh, 1970, pp 199-206.

A preferred binding partner for these assays is an anti-immunoglobulinantibody (“second antibody”) produced in a different species. Thus todetect a nonhuman primate antibody, a detectably labeled goatanti-simian immunoglobulin “second” antibody may be used. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on the solid support maythen be detected by conventional means appropriate to the type of labelused (see below).

Such a “second antibody” may be specific for epitopes characteristic ofa particular human immunoglobulin isotype, for example IgM, IgG₁,IgG_(2a), IgA and the like, thus permitting identification of theisotype or isotypes of antibodies in the sample which are specific forthe Mtb antigen. Alternatively, the second antibody may be specific foran idiotype of the anti-Mtb antibody of the sample.

As alternative binding partners for detection of the sample antibody,other known binding partners for human immunoglobulins may be used.Examples are the staphylococcal immunoglobulin binding proteins, thebest know of which is protein A. Also intended is staphylococcal proteinG, or a recombinant fusion protein between protein A and protein G.Protein G of group G and group C streptococci binds to the Fc portion ofIg molecules as well as to IgG Fab fragment at the V_(H3) domain.Protein C of Peptococcus magnus binds to the Fab region of theimmunoglobulin molecule. Any other microbial immunoglobulin bindingproteins, for example from Streptococci, are also intended (for example,Langone, J. J., Adv. Immunol 32:157 (1982)).

In another embodiment of this invention, a biological fluid suspected ofcontaining antibodies specific for Mtb antigens may be brought intocontact with a solid support or carrier which is capable of immobilizingsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with Mtb antigens reagent, which may be detectablylabeled. Bound antigen is then measured by measuring the immobilizeddetectable label. If the Mtb antigen reagent is not directly detectablylabeled, a second reagent comprising a detectably labeled bindingpartner for the Mtb antigen, generally a second anti-Mtb antibody suchas a murine mAb, is allowed to bind to any immobilized antigen. Thesolid phase support may then be washed with buffer a second time toremove unbound antibody. The amount of bound label on said solid supportmay then be detected by conventional means.

By “solid phase support” or carrier is intended any support capable ofbinding a proteinaceous antigen or antibody molecules or other bindingpartners according to the present invention. Well-known supports, orcarriers, include glass, polystyrene, polypropylene, polyethylene,polyvinylidene difluoride, dextran, nylon, magnetic beads, amylases,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as it is capable of binding to an antigen orantibody. Thus, the support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube, or theexternal surface of a rod. Alternatively, the surface may be flat suchas a sheet, test strip, etc. Preferred supports include polystyrenebeads, 96-well polystyrene microplates and test strips, all well-knownin the art. Those skilled in the art will know many other suitablecarriers for binding antibody or antigen, or will be able to ascertainthe same by use of routine experimentation.

Using any of the assays described herein, those skilled in the art willbe able to determine operative and optimal assay conditions for eachdetermination by employing routine experimentation. Furthermore, othersteps as washing, stirring, shaking, filtering and the like may be addedto the assays as is customary or necessary for the particular situation.

In some embodiments, the present invention provides protein chip assayscomprising one or more capture reagents or antigenic compositionscomprising at least one of SEQ ID NOs:1-149. In such an assay, thecapture reagents or antigenic compositions are immobilized on a solidsupport such as a chip. In some embodiments, the protein chip assayutilizes a solid support coated with ultrathin or clear nitrocellulose.See, e.g., US PAT PUBL. 20090253586 and 20090075828, both of which areincorporated herein by reference in their entirety. In preferredembodiments, the capture reagents or antigenic compositions are arrayedon the solid support. In multiplexed assays, a panel of capture reagentsor antigenic compositions as described above is arrayed on the solidsupport. See, e.g., US PAT PUBL. 20090253586 and 20090075828, both ofwhich are incorporated herein by reference in their entirety. A samplefrom a subject is passed over the solid support. Bound antibodies fromthe sample are then detected using any suitable method. Other suitableprotein chip assays are described, for example, in U.S. Pat. No.6,197,599; U.S. Pat. No. 6,294,790 and US Patent ApplicationUS20010014461A1, each of which is herein incorporated by reference inits entirety).

In some embodiments, a cytometric bead array assay is used (Quantum Plexkit, Bangs Laboratories; Cytometric Bead Array kit, BD Biosciences).These systems allow for multiple analyte detection with small volumesamples. In other embodiments, a LUMINEX bead assay is used. See, e.g.,U.S. Pat. Nos. 6,916,661; 6,939,720; 7,141,431; 7,445,844; 7,465,540;8,038,734; and 8,088,629, all of which are incorporated herein byreference in their entirety.

In some embodiments, the immunoassay used to detect an antibody specificfor an Mtb antigen according to the present invention is anenzyme-linked immunosorbent assay (ELISA) or more generically termed anenzyme immunoassay (EIA). In such assays, a detectable label bound toeither an antibody-binding or antigen-binding reagent is an enzyme. Whenexposed to its substrate, this enzyme will react in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label the reagents useful in the present inventioninclude, but are not limited to, horseradish peroxidase, alkalinephosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease,catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase,delta-5-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. For descriptions of EIA procedures, see referencecited above or, additionally, Voller, A. et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980.

In some embodiments, the immunoassay devices of the present inventionpermit the performance of relatively inexpensive, disposable,membrane-based assays for the visual identification of the presence (orabsence) of an analyte in a liquid sample. Such devices are usuallyformatted as freestanding dipsticks (e.g., test strips) or as deviceshaving some sort of housing. Typically, an immunoassay device of thepresent invention can be used with as little as about 200 μl of liquidsample, and detection of an analyte in the sample can (but need not) becomplete within 2-5 minutes. In preferred embodiments, no ancillaryinstrumentation is required to perform such tests, and such deviceseasily can be used in clinics, laboratories, field locations, and thehome even by inexperienced persons.

In some embodiments, the ELISA is an immunochromatographic “sandwich”assay. In general, sandwich immunochromatographic procedures call formixing the sample that may contain the analyte to be assayed, forexample, Mtb antibodies, with an antigenic composition or capturereagent as described above. A detector reagent is utilized which ismobile and typically is linked to a label or another signaling reagent,such as dyed latex, a colloidal metal sol, or a radioisotope. Thismixture is then applied to a chromatographic medium containing a band orzone of immobilized antigenic compositions that serve as antigens forMtb antibodies (i.e., the capture reagent). The chromatographic mediumoften is in the form of a strip that resembles a dipstick. When thecomplex of Mtb antibody and the detector reagent reaches the zone of theimmobilized capture antibody on the chromatographic medium, bindingoccurs and the detector reagent complex is localized at the zone. Thisindicates the presence of the molecule to be assayed. This technique canbe used to obtain quantitative or semi-quantitative results. Examples ofsandwich immunoassays performed on test strips are described in U.S.Pat. Nos. 4,168,146 and 4,366,241, each of which is incorporated hereinby reference.

In other embodiments, the ELISA is a solid phase immunoassay device thatprovides sensitive detection of analytes in biological fluid samples.Solid phase immunoassay devices incorporate a solid support to which onemember of a ligand-receptor pair, usually an antibody, antigen, orhapten, is bound. Common early forms of solid supports were plates,tubes, or beads of polystyrene, which were known from the fields ofradioimmunoassay and enzyme immunoassay. More recently, a number ofporous materials such as nylon, nitrocellulose, cellulose acetate, glassfibers, and other porous polymers have been employed as solid supports.In other common forms of membrane-based immunoassays, as typified bysome home pregnancy and ovulation detection kits, a test strip (ordipstick) is “dipped” into a sample suspected of containing the subjectanalyte. Enzyme-labeled detector reagent is then added, eithersimultaneously or after an incubation period. The device next is washedand then inserted into a second solution containing a substrate for theenzyme. The enzyme label, if present, interacts with the substrate,causing the formation of colored products, which either deposit as aprecipitate onto the solid phase or produce a visible color change inthe substrate solution. EP-A 0 125 118 describes such a sandwich typedipstick immunoassay. EP-A 0 282 192 describes a dipstick device for usein competition type assays.

In other embodiments, the assay device of the present invention is aflow through immunoassay device. Flow-through immunoassay devicesinvolve a capture reagent (such as an immunogenic composition comprisingat least of SEQ ID NOs:1-149) bound to a porous membrane or filter towhich a liquid sample is added. As the liquid flows through themembrane, target analyte (such as an Mtb antibody) binds to the capturereagent. The addition of sample is followed by (or made concurrent with)addition of detector reagent (e.g., labeled second antibody).Alternatively, the detector reagent may be placed on the membrane in amanner that permits the detector to mix with the sample and therebylabel the analyte. The visual detection of detector reagent provides anindication of the presence of target analyte in the sample.Representative flow-through immunoassay devices are described in U.S.Pat. Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and5,279,935; and U.S. Patent Application Publication Nos. 20030049857 and20040241876, all of which are incorporated by reference in theirentirety. In some embodiments, the assay device is a migration assaydevice. Such devices usually incorporate within them reagents that havebeen attached to colored labels, thereby permitting visible detection ofthe assay results without addition of further substances. See, forexample, U.S. Pat. No. 4,770,853; PCT Publication No. WO 88/08534 andEuropean Patent No. EP-A 0 299 428, all of which are incorporated byreference in their entirety.

In some embodiments, the assay device is lateral flow assay device.There are a number of commercially available lateral flow type tests andpatents disclosing methods for the detection of analytes. See, e.g.,U.S. Pat. Nos. 5,229,073; 5,591,645; 4,168,146; 4,366,241; 4,855,240;4,861,711; 4,703,017; 5,451,504; 5,451,507; 5,798,273; 6,001,658; and5,120,643; European Patent No. 0296724; WO 97/06439; and WO 98/36278,all of which are incorporated herein by reference.

The lateral flow assay devices of the present invention include a stripof absorbent or porous material (such as a microporous membrane), which,in some instances, can be made of different substances each joined tothe other in zones, which may be abutted and/or overlapped. In someexamples, the absorbent strip can be fixed on a supportingnon-interactive material (such as nonwoven polyester), for example, toprovide increased rigidity to the strip. Zones within each strip maydifferentially contain the specific binding partner(s) and/or otherreagents required for the detection and/or quantification of theparticular analyte being tested for, for example, Mtb antibodies. Thusthese zones can be viewed as functional sectors or functional regionswithin the test device.

In some embodiments, a fluid sample (or a sample suspended in a fluid)is introduced to the strip at the proximal end of the strip, forinstance by dipping or spotting. A sample is collected or obtained usingmethods well known to those skilled in the art. The sample containingthe Mtb antibodies to be detected may be obtained from any biologicalsource. Examples of biological sources include blood serum, bloodplasma, urine, spinal fluid, saliva, fermentation fluid, lymph fluid,tissue culture fluid and ascites fluid of a human or animal. The samplemay be diluted, purified, concentrated, filtered, dissolved, suspendedor otherwise manipulated prior to immunoassay to optimize theimmunoassay results. The fluid migrates distally through all thefunctional regions of the strip. The final distribution of the fluid inthe individual functional regions depends on the adsorptive capacity andthe dimensions of the materials used.

In some embodiments, porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

In some embodiments, the assay devices include a detector reagent. Thedetector reagent provides a means to detect the formation of a complexbetween an analyte (such as an Mtb antibody or antibodies) and a capturereagent (such as an antigenic composition as described above). Adetector may be integrated into an immunoassay device (for exampleincluded in a conjugate pad, as described below), or may be applied tothe device from an external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte. In otherinstances, a detector reagent collectively includes an unlabeled firstbinding partner specific for the analyte and a labeled second bindingpartner specific for the first binding partner and so forth. In eachinstance, a detector reagent specifically detects bound analyte of ananalyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent can specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

The flow-through devices of the present invention comprise a capturereagent (e.g., antigenic composition as described above) immobilized ona solid support such as a microtiter plate or a membrane (such as,nitrocellulose, nylon, or PVDF). Characteristics of useful membrane havebeen previously described; however, it is useful to note that in aflow-through assay capillary rise is not a particularly importantfeature of a membrane as the sample moves vertically through themembrane rather than across it as in a lateral flow assay. In a simplerepresentative format, the membrane of a flow-through device is placedin functional or physical contact with an absorbent layer (see, e.g.,description of “absorbent pad” below), which acts as a reservoir to drawa fluid sample through the membrane. Optionally, followingimmobilization of a capture reagent, any remaining protein-binding siteson the membrane can be blocked (either before or concurrent with sampleadministration) to minimize nonspecific interactions.

In operation of a flow-through device, a fluid sample (such as a bodilyfluid sample) is placed in contact with the membrane. Typically, aflow-through device also includes a sample application area (orreservoir) to receive and temporarily retain a fluid sample of a desiredvolume. The sample passes through the membrane matrix. In this process,an analyte in the sample (e.g., Mtb antibody or antibodies) canspecifically bind to the immobilized capture reagent. Where detection ofan analyte-capture reagent complex is desired, a detector reagent (e.g.,labeled Protein A, labeled second antibody) can be added with the sampleor a solution containing a detector reagent can be added subsequent toapplication of the sample.

If an analyte is specifically bound by capture reagent, a visualrepresentative attributable to the particular detector reagent can beobserved on the surface of the membrane. Optional wash steps can beadded at any time in the process, for instance, following application ofthe sample, and/or following application of a detector reagent. Alateral flow device is an analytical device comprising a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte canflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte. Manylateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials can be used, and rendered bibulous, e.g., byapplying a surfactant to the material), and allowed to migrate along thestrip until the liquid comes into contact with a specific bindingpartner that interacts with an analyte in the liquid. Once the analyteinteracts with the binding partner, a signal (such as a fluorescent orotherwise visible dye) indicates that the interaction has occurred.Multiple discrete binding partners can be placed on the strip (forexample in parallel lines) to detect multiple analytes in the liquid.The test strips can also incorporate control indicators, which provide asignal that the test has adequately been performed, even if a positivesignal indicating the presence (or absence) of an analyte is not seen onthe strip.

The construction and design of lateral flow devices is described, forexample, in Millipore Corporation, A Short Guide DevelopingImmunochromatographic Test Strips, 2nd Edition, pp. 1-40, 1999,available by request at (800) 645-5476; and Schleicher & Schuell, Easyto Work with BioScience, Products and Protocols 2003, pp. 73-98, 2003,available by request at Schleicher & Schuell BioScience, Inc., 10Optical Avenue, Keene, N.H. 03431, (603) 352-3810; both of which areincorporated herein by reference. Lateral flow devices have a widevariety of physical formats. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure.

In some embodiments, lateral flow devices of the present inventioncomprise an elongated housing containing a bibulous lateral flow stripthat extends substantially the entire length of housing. In someembodiments, the lateral flow strip is divided into a proximal sampleapplication pad positioned below a sample introduction port, anintermediate test result membrane, and a distal absorbent pad. The flowstrip is interrupted by a conjugate pad that contains labeled conjugate(such labeled second antibody). A flow path along the strip passes fromthe proximal pad, through conjugate pad, into a test result membrane,for eventual collection in absorbent pad. Selective binding agents (suchas the antigenic compositions described above) are positioned on aproximal test line in the test result membrane. A control line isprovided in the test result membrane slightly distal to the test line. Afluid sample containing an analyte of interest, such as Mtb antibody orantibodies, is applied to the sample pad through the sample introductionport. In some embodiments, the sample may be applied to the sampleintroduction port dropwise or by dipping the end of the devicecontaining the sample introduction port into the sample. From the samplepad, the sample passes, for instance by capillary action, to theconjugate pad. In the conjugate pad, the analyte of interest may bind(or be bound by) a mobilized or mobilizable detector reagent. Forexample, an Mtb antibody may bind to a labeled (e.g., gold-conjugated)detector reagent (such as a second antibody contained in the conjugatepad. The analyte complexed with the detector reagent may subsequentlyflow to the test result membrane where the complex may further interactwith a capture reagent, such as an antigenic composition as describedabove, which is immobilized at the proximal test line. The formation ofthe immunocomplex between Mtb antibody, labeled (e.g., gold-conjugated)detector reagent, and immobilized antigenic composition can be detectedby the appearance of a visible line at the proximal test line, whichresults from the accumulation of the label (e.g., gold) in the localizedregion of the proximal test line. The control line may contain animmobilized, detector-reagent-specific binding partner, which can bindthe detector reagent in the presence or absence of the analyte. Suchbinding at the control line indicates proper performance of the test,even in the absence of the analyte of interest.

The particular materials used in a particular lateral flow device willdepend on a number of variables, including, for example, the analyte tobe detected, the sample volume, the desired flow rate and others. Insome embodiments, the sample pad receives the sample, and may serve toremove particulates from the sample. In some embodiments, the sample padis cellulose. Sample pads may be treated with one or more releaseagents, such as buffers, salts, proteins, detergents, and surfactants.Such release agents may be useful, for example, to promoteresolubilization of conjugate-pad constituents, and to blocknon-specific binding sites in other components of a lateral flow device,such as a nitrocellulose membrane. Representative release agentsinclude, for example, trehalose or glucose (1%-5%), PVP or PVA(0.5%-2%), Tween 20 or Triton X-100 (0.1%-1%), casein (1%-2%), SDS(0.02%-5%), and PEG (0.02%-5%).

The conjugate pad holds a detector reagent. In some embodiments, adetector reagent may be applied externally, for example, from adeveloper bottle, in which case a lateral flow device need not contain aconjugate pad (see, for example, U.S. Pat. No. 4,740,468). Detectorreagent(s) contained in a conjugate pad is typically released intosolution upon application of the test sample. A conjugate pad may betreated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose.

The absorbent pad acts to increase the total volume of sample thatenters the device. This increased volume can be useful, for example, towash away unbound analyte from the membrane. Any of a variety ofmaterials is useful to prepare an absorbent pad. In some deviceembodiments, an absorbent pad can be paper (i.e., cellulosic fibers).One of skill in the art may select a paper absorbent pad on the basisof, for example, its thickness, compressibility, manufacturability, anduniformity of bed volume. The volume uptake of an absorbent made may beadjusted by changing the dimensions (usually the length) of an absorbentpad.

A wide variety of detectable labels are useful with the assays describedabove in addition to the described enzymatic labels.

In another embodiment, the detectable label may be a gold or silvernanoparticle that can be enhanced with non-enzymatic silver deposition(SilverQuant™). Methods for detection with silver or gold nanoparticlesare described in detail in U.S. Pat. No. 7,321,829, incorporated byreference herein its entirety, as well as in US PUBL. 20090253586, alsoincorporated herein by reference in its entirety.

In another embodiment, the detectable label may be a Proximity LigationAssay (PLA) reagent[25]. Proximity ligation assay (PLA) is an approachfor protein quantitation that can use two different binder molecules(proximity probes) to bind to a specific detection target (See forexample Fredriksson, S. et al., Nat. Biotechnol. 2002; 20(5): 473-77,Gullberg, M., et. al., Proc Natl Acad Sci USA. 2004; 101(22): 8420-24,Gullberg, M., et. al., Curr Opin Biotechnol. 2003; 14: 1-5, Pai, S.,Ellington, A. D. and Levy, M., Nuc Acids Res. Oct. 19 2005; 33(18):e162, Landegren, U. and Fredriksson, S., US Patent Application20020064779, May 30, 2002, Fredriksson, S., US Patent Application20050003361, all pof which are incorporated by reference herein in theirentirety. Typical binders include polyclonal or monoclonal antibodypairs. Each binder molecule can be conjugated to a specificoligonucleotide. One binder's oligonucleotide can form the “left” sideof a real-time PCR amplicon, while the other binder can form the “right”side. When the two binders find and attach to the same target, the leftand right oligomers are brought into close proximity. With the additionof a connector oligonucleotide (splint) and ligase enzyme, the left andright oligomers can become ligated and thereby allow for the formationof a complete target for a real-time PCR. Further addition of Taqmanreaction components followed by thermocycling generates real-timesequence detection data output.

In another embodiment, the detectable label may be a radiolabel, and theassay termed a radioimmunoassay (RIA), as is well known in the art. Theradioisotope can be detected by a gamma counter, a scintillation counteror by autoradiography. Isotopes which are particularly useful for thepurpose of the present invention are ¹²⁵I, ¹³¹I, ³⁵S, ³H and ¹⁴C.

It is also possible to label the antigen or antibody reagents with afluorophore. When the fluorescently labeled antibody is exposed to lightof the proper wave length, its presence can then be detected due tofluorescence of the fluorophore. Among the most commonly usedfluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine orfluorescence-emitting metals such as ¹⁵²Eu or other lanthanides. Thesemetals are attached to antibodies using metal chelators.

The antigen or antibody reagents useful in the present invention alsocan be detectably labeled by coupling to a chemiluminescent compound.The presence of a chemiluminescent-tagged antibody or antigen is thendetermined by detecting the luminescence that arises during the courseof a chemical reaction. Examples of useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescentcompound such as a bioluminescent protein may be used to label theantigen or antibody reagent useful in the present invention. Binding ismeasured by detecting the luminescence. Useful bioluminescent compoundsinclude luciferin, luciferase and aequorin.

Detection of the detectably labeled reagent according to the presentinvention may be accomplished by a scintillation counter, for example,if the detectable label is a radioactive gamma emitter, or by afluorometer, for example, if the label is a fluorophore. In the case ofan enzyme label, the detection is accomplished by colorimetry to measurethe colored product produced by conversion of a chromogenic substrate bythe enzyme. Detection may also be accomplished by visual comparison ofthe colored product of the enzymatic reaction in comparison withappropriate standards or controls.

In some embodiments, the one or more of the peptides or conjugatesdescribed above (alone or in combination) are used as an antigenstimulation mixture for cell based assays including, but not limited to,cytokine release assays (particularly interferon gamma release andinterleukin 12) as measured by ELISA, Elispot, or bead based methods. Inother embodiments, the peptides or conjugates described above (alone orin combination) are used in T-cell capture assays. In still otherembodiments, the peptides or conjugates described above (alone or incombination) are used as an antigenic substitute for tuberculin in thetuberculin skin test (TST).

In some embodiments, the present invention provides kits for use indetecting Mtb antibodies in a sample (such as, a biological sample).Such kits can be used, for example, to determine whether an animal hasbeen exposed to Mtb or has an Mtb infection. Certain embodiments of thedisclosed kits are generally portable and provide a simple, rapid,and/or cost-effective way to determine Mtb infection without the needfor specialized laboratory facilities, such as in a point-of-carefacility.

In some embodiments, the kits of the present invention include one ormore immunoassay devices (and/or antigen-coated microtiter plates) asdisclosed herein and a carrier means, such as a box, a bag, a satchel,plastic carton (such as molded plastic or other clear packaging),wrapper (such as, a sealed or sealable plastic, paper, or metallicwrapper), or other container. In some examples, kit components will beenclosed in a single packaging unit, such as a box or other container,which packaging unit may have compartments into which one or morecomponents of the kit can be placed. In other examples, a kit includesone or more containers, for instance vials, tubes, and the like that canretain, for example, one or more biological samples to be tested,positive and/or negative control samples or solutions (such as, apositive control serum containing Mtb antibodies), diluents (such as,phosphate buffers, or saline buffers), detector reagents (e.g., forexternal application to a kit device), substrate reagents forvisualization of detector reagent enzymes (such as,5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium in dimethylformamide), and/or wash solutions (such as, Tris buffers, saline buffer,or distilled water).

Other kit embodiments include syringes, finger-prick devices, alcoholswabs, gauze squares, cotton balls, bandages, latex gloves, incubationtrays with variable numbers of troughs, adhesive plate sealers, datareporting sheets, which may be useful for handling, collecting and/orprocessing a biological sample. Kits may also optionally containimplements useful for introducing samples into a sample chamber of animmunoassay device, including, for example, droppers, Dispo-pipettes,capillary tubes, rubber bulbs (e.g., for capillary tubes), and the like.Still other kit embodiments may include disposal means for discarding aused immunoassay device and/or other items used with the device (such aspatient samples, etc.). Such disposal means can include, withoutlimitation, containers that are capable of containing leakage fromdiscarded materials, such as plastic, metal or other impermeable bags,boxes or containers.

In some embodiments, a kit of the present invention will includeinstructions for the use of an immunoassay device or antigen-coatedplate. The instructions may provide direction on how to apply sample tothe test device or plate, the amount of time necessary or advisable towait for results to develop, and details on how to read and interpretthe results of the test. Such instructions may also include standards,such as standard tables, graphs, or pictures for comparison of theresults of a test. These standards may optionally include theinformation necessary to quantify analyte using the test device, such asa standard curve relating intensity of signal or number of signal linesto an amount of analyte therefore present in the sample.

In some embodiments, the present invention provides a vaccine comprisingone or more of the peptides described above (i.e. SEQ ID NOs.:1-149). Asused herein, the term “vaccine” refers to any combination of peptides ora single peptide formulation. There are various reasons why one mightwish to administer a vaccine of a combination of the peptides of thepresent invention rather than a single peptide. Depending on theparticular peptide that one uses, a vaccine might have superiorcharacteristics as far as clinical efficacy, solubility, absorption,stability, toxicity and patient acceptability are concerned. It shouldbe readily apparent to one of ordinary skill in the art how one canformulate a vaccine of any of a number of combinations of peptides ofthe present invention. There are many strategies for doing so, any oneof which may be implemented by routine experimentation.

The peptides of the present invention may be administered as a singleagent therapy or in addition to an established therapy, such asinoculation with live, attenuated, or killed virus, or any other therapyknown in the art to treat the target disease or epitope-sensitivecondition.

The appropriate dosage of the peptides of the invention may depend on avariety of factors. Such factors may include, but are in no way limitedto, a patient's physical characteristics (e.g., age, weight, sex),whether the compound is being used as single agent or adjuvant therapy,the type of MHC restriction of the patient, the progression (i.e.,pathological state) of the infection or other epitope-sensitivecondition, and other factors that may be recognized by one skilled inthe art. In general, an epitope or combination of epitopes may beadministered to a patient in an amount of from about 50 micrograms toabout 5 mg; dosage in an amount of from about 50 micrograms to about 500micrograms is especially preferred.

In some embodiments, the peptides are expressed on bacteria, such aslactococcus and lactobacillus, or expressed on virus or virus-likeparticles for use as vaccines. In some embodiments, the peptides areincorporated into other carriers as are known in the art. For example,in some embodiments, the polypeptides comprising one or more epitopesare conjugated or otherwise attached to a carrier protein. Suitablecarrier proteins include, but are not limited to keyhole limpethemocyanin, bovine serum albumin, ovalbumin, and thyroglobulin. In yetother embodiments the polypeptide may be fused to an Fc region of animmunoglobulin for delivery to a mucosal site bearing correspondingreceptors.

One may administer a vaccine of the present invention by any suitablemethod, which may include, but is not limited to, systemic injections(e.g., subcutaneous injection, intradermal injection, intramuscularinjection, intravenous infusion) mucosal administrations (e.g., nasal,ocular, oral, vaginal and anal formulations), topical administration(e.g., patch delivery), or by any other pharmacologically appropriatetechnique. Vaccination protocols using a spray, drop, aerosol, gel orsweet formulation are particularly attractive and may be also used. Thevaccine may be administered for delivery at a particular time interval,or may be suitable for a single administration.

Vaccines of the invention may be prepared by combining at least onepeptide with a pharmaceutically acceptable liquid carrier, a finelydivided solid carrier, or both. As used herein, “pharmaceuticallyacceptable carrier” refers to a carrier that is compatible with theother ingredients of the formulation and is not toxic to the subjects towhom it is administered. Suitable such carriers may include, forexample, water, alcohols, natural or hardened oils and waxes, calciumand sodium carbonates, calcium phosphate, kaolin, talc, lactose,combinations thereof and any other suitable carrier as will berecognized by one of skill in the art. In a most preferred embodiment,the carrier is present in an amount of from about 10 uL (micro-Liter) toabout 100 uL.

In some embodiments, the vaccine composition includes an adjuvant.Examples of adjuvants include, but are not limited to, mineral salts(e.g., aluminum hydroxide and aluminum or calcium phosphate gels); oilemulsions and surfactant based formulations (e.g., MF59 (microfluidizeddetergent stabilized oil-in-water emulsion), QS21 (purified saponin),Ribi Adjuvant Systems, AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),Montanide ISA-51 and ISA-720 (stabilized water-in-oil emulsion);particulate adjuvants (e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), AS04 ([SBAS4] A1 salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidalimmunostimulators able to self organize into liposomes), OM-174 (lipid Aderivative), CpG motifs (synthetic oligonucleotides containingimmunostimulatory CpG motifs), modified LT and CT (genetically modifiedbacterial toxins to provide non-toxic adjuvant effects); endogenoushuman immunomodulators (e.g., hGM-CSF or hIL-12 (cytokines that can beadministered either as protein or plasmid encoded), Immudaptin (C3dtandem array); and inert vehicles, such as gold particles. In variousembodiments, vaccines according to the invention may be combined withone or more additional components that are typical of pharmaceuticalformulations such as vaccines, and can be identified and incorporatedinto the compositions of the present invention by routineexperimentation. Such additional components may include, but are in noway limited to, excipients such as the following: preservatives, such asethyl-p-hydroxybenzoate; suspending agents such as methyl cellulose,tragacanth, and sodium alginate; wetting agents such as lecithin,polyoxyethylene stearate, and polyoxyethylene sorbitan mono-oleate;granulating and disintegrating agents such as starch and alginic acid;binding agents such as starch, gelatin, and acacia; lubricating agentssuch as magnesium stearate, stearic acid, and talc; flavoring andcoloring agents; and any other excipient conventionally added topharmaceutical formulations.

Further, in various embodiments, vaccines according to the invention maybe combined with one or more of the group consisting of a vehicle, anadditive, a pharmaceutical adjunct, a therapeutic compound or agentuseful in the treatment of the desired disease, and combinationsthereof.

EXAMPLES Example 1 Material and Methods

Peptides

A partial proteome mapping peptide array of 6912 peptides 15 amino acidsin length with a 12 amino acid overlap (product RT-HD-TUB) for thereference strain Mycobacterium tuberculosis H37Rv (JPT PeptideTechnologies, GmbH, Berlin, Germany) was used to perform preliminarystudies. A refined set of 1075 peptides were designed for proteins ofinterest from these experiments, covering 246 proteins, with 15 aminoacid length with a 3 amino acid overlap.

Array Preparation

Arrays were manufactured with peptides synthesized in the mannerdescribed in Nahtman et al[27]. Briefly, 1 mM solution of each peptidewas printed on epoxy functionalized glass slides (Corning Epoxy #440042)using a contact array printer (JPT Peptide). Arrays were printed suchthat each peptide was printed in triplicate. For the refined set of 1075peptides, arrays were printed with 4 subarrays per slide, withtriplicate spots. Human IgG was printed as internal controls for theassay, and orientation for the data analysis.

Serum Samples

Preliminary experiments were performed with samples generously donatedby the Pathogen Detection Laboratory at the California National PrimateCenter (University of California-Davis, Davis, Calif.). Six serumsamples were used for the preliminary studies, including 4 time pointsduring a course of an intentional experimental infection, and serum from2 known test positive macaques.

Samples for assays performed on the refined arrays were generouslydonated by the Oregon National Primate Research Center (Oregon Healthand Science University, Beaverton, Oreg.). Sera from twelve animalsquarantined and necropsied following an outbreak of M. tb in the colonywere compared with sera from previous blood collections, 1-2 years priorto the outbreak. In addition to pre-infection sera, 4 test negativesamples from the same colony were also run on the refined arrays toestablish an average “non-infected” immune profile. Additionally, 56test negative samples from the Wisconsin National Primate Center (giftof Dr. Jonah Sacha) and the Caribbean National Primate Center (gift ofDr. Carlos Sariol) were also included in the negative serum set. Table 1shows the individual serum samples, their source, and previous testresults for tuberculin skin test (TST) and other confirmatory testing.Sera were stored at −80° C. until use.

TABLE 1 Sample Sample Disease ID Source Type State TST Test 17846 WNPRCNormal Naïve Negative 17847 WNPRC Normal Naïve Negative 17848 WNPRCNormal Naïve Negative 17849 WNPRC Normal Naïve Negative 17850 WNPRCNormal Naïve Negative 17851 WNPRC Normal Naïve Negative 17852 WNPRCNormal Naïve Negative 17853 WNPRC Normal Naïve Negative 17854 WNPRCNormal Naïve Negative 17855 WNPRC Normal Naïve Negative 17856 WNPRCNormal Naïve Negative 17857 WNPRC Normal Naïve Negative 17858 WNPRCNormal Naïve Negative 17859 WNPRC Normal Naïve Negative 17860 WNPRCNormal Naïve Negative 17861 WNPRC Normal Naïve Negative 17862 WNPRCNormal Naïve Negative 17863 WNPRC Normal Naïve Negative 17864 WNPRCNormal Naïve Negative 17865 WNPRC Normal Naïve Negative 17866 WNPRCNormal Naïve Negative 17867 WNPRC Normal Naïve Negative 17868 WNPRCNormal Naïve Negative 17869 WNPRC Normal Naïve Negative 20758 ONPRCDisease Post Positive 20284 ONPRC Disease Post Positive 22319 ONPRCDisease Post Positive 28642 ONPRC Disease Post Positive 28643 ONPRCDisease Post Positive 28655 ONPRC Disease Post Positive 28667 ONPRCDisease Post Positive 28674 ONPRC Disease Post Positive 28676 ONPRCDisease Post Positive 28677 ONPRC Disease Post Positive 29417 ONPRCDisease Post Positive 20758 ONPRC Normal Pre Negative 20284 ONPRC NormalPre Negative 22319 ONPRC Normal Pre Negative 28642 ONPRC Normal PreNegative 28643 ONPRC Normal Pre Negative 28655 ONPRC Normal Pre Negative28667 ONPRC Normal Pre Negative 28674 ONPRC Normal Pre Negative 28676ONPRC Normal Pre Negative 28677 ONPRC Normal Pre Negative 29417 ONPRCNormal Pre Negative 27790 ONPRC Normal Naïve Negative 26023 ONPRC NormalNaïve Negative 22819 ONPRC Normal Naïve Negative 23351 ONPRC NormalNaïve Negative 18171 CaNPRC Normal Naïve Negative 18172 CaNPRC NormalNaïve Negative 18173 CaNPRC Normal Naïve Negative 18174 CaNPRC NormalNaïve Negative 18175 CaNPRC Normal Naïve Negative 18176 CaNPRC NormalNaïve Negative 18177 CaNPRC Normal Naïve Negative 18178 CaNPRC NormalNaïve Negative 18179 CaNPRC Normal Naïve Negative 18180 CaNPRC NormalNaïve Negative 18181 CaNPRC Normal Naïve Negative 18182 CaNPRC NormalNaïve Negative 18183 CaNPRC Normal Naïve Negative 18184 CaNPRC NormalNaïve Negative 18185 CaNPRC Normal Naïve Negative 18186 CaNPRC NormalNaïve Negative 18187 CaNPRC Normal Naïve Negative 18188 CaNPRC NormalNaïve Negative 18189 CaNPRC Normal Naïve Negative 18190 CaNPRC NormalNaïve Negative 18191 CaNPRC Normal Naïve Negative 18192 CaNPRC NormalNaïve Negative 18193 CaNPRC Normal Naïve Negative 18194 CaNPRC NormalNaïve Negative 18195 CaNPRC Normal Naïve Negative 18196 CaNPRC NormalNaïve Negative 18197 CaNPRC Normal Naïve Negative 18198 CaNPRC NormalNaïve Negative 18199 CaNPRC Normal Naïve Negative 18200 CaNPRC NormalNaïve Negative 18201 CaNPRC Normal Naïve Negative 18202 CaNPRC NormalNaïve Negative Key Naïve Normal, uninfected animal Pre Normal samplefrom animal that later was diagnosed with TB Post Disease sample fromanimal diagnosed with TB ONPRC Oregon National Primate Center WNPRCWisconsin National Primate Center CaNRPC Caribbean National PrimateCenterSample Processing

A reusable frame and gasket system (Intuitive Biosciences, Madison,Wis.) for incubation was applied to the peptide microarrays. Serum wasdiluted 1:100 in Dilution Buffer (Intuitive Biosciences), and incubatedin one well of the array. Detection of bound IgG from the serum sampleswas performed using the CSA: Simian Detection kit (IntuitiveBiosciences). Briefly, diluted serum was incubated for 1 hr at roomtemperature. Each well was washed 5 times, followed by addition of abiotinylated anti-simian IgG antibody diluted 1:10,000, and incubatedfor 1 hour at room temperature. After 5 repeat washes, 1 mL of goldconjugated anti-biotin was incubated for 45 minutes at room temperature.After 5 wash steps, slides were rinsed with 1 mL of water and immunecomplexes visualized by addition of SilverQuant® reagents (IntuitiveBiosciences). After 3 minutes incubation in dark, slides were quicklywashed with water and dried.

Data Acquisition

The peptide microarray slides were scanned with the AthenaQuant Systemfrom Intuitive Biosciences. Saved images were analyzed with AthenaQuant®software from Intuitive Biosciences, and background subtracted dataexported in Excel files. Relative Intensity Units (RIU) were calculatedby AthenaQuant and reported for each peptide feature on the microarray.

Statistical Analysis

Preliminary studies used RIU data from the mean of triplicate spots foreach sample to calculate a fold over normal value.

The majority of the data management and analysis steps were accomplishedwith the Golden Helix SNP & Variation Suite (SVS) software version 7.5(Golden Helix, Boseman, Mont.). Data was normalized by two methods:quantile normalization and Principal Components Analysis. Comparisons ofinfected and normal samples were conducted using two statistical tests:a Two-Sample T-Test and a Mann-Whitney (MW) Rank Sum Test. Two t-testsand two MW tests were performed. Each test was performed for thecomparison of the matched pair (pre infection vs post infection) dataand the comparison of the 12 TB-positive samples to the 60 naivesamples. The matched-pair tests were performed only inquantile-normalized data, while the 12 positive vs 60 naive tests wereperformed in the PCA-corrected data. The resulting p-values for eachcomparison were reported in Excel, and a sum of the p-values for thefour tests were calculated and used to sort the peptides in order ofsignificance.

Results

Samples from the Oregon National Primate Center (ONPRC) were acquiredfollowing a series of positive TST during routine screening. One animalpresented a positive TST, resulting in the immediate quarantine of thatanimal and the other housed in the same room, a total of twelve animals.Several tests were performed in parallel with repeated TST on thequarantined animals. Of the 12 animals, 7 tested positive by TSTperformed at the ONPRC. A PrimaTB STAT-Pak, referred to as Lateral Flowin Table 2, was performed according to the manufacturer's instructionsby the ONPRC. Whole blood samples were collected from these animals andsent to the Pathogen Detection Laboratory at UC-Davis (PDL), where thePrimagam assay for measuring IFN-γ release was performed alongside anESAT-6 ELISA for detection of ESAT-6 specific antibodies. At least 7animals tested positive by these methods, and the animals wereeuthanized and post-mortem testing performed. Mycobacterium culture wasperformed from lung samples by the National Veterinary ServicesLaboratories (Ames, Iowa) and found 8 samples culture positive.Additionally, PCR testing for detection of Mycobacteria from the sampleswas performed by the Washington Animal Disease Diagnostic Lab (WADDL)but only found a few positive samples. Lastly, gross pathology of lungswas performed during necropsy of the animals and lesions were found inten of the twelve animals. Serum samples collected from these twelveanimals during quarantine were received along with serum samples fromblood draws at least 2 years prior to the outbreak, when the animals hadnegative TST. These pre-infection and post-infection samples were usedin addition to the other naïve samples listed in Table 1 to performantibody detection assays using the refined TB peptide microarrays.Statistical analysis of the 12 positive sera compared to the 60 naïvesera was performed, and a set of 5 peptides were chosen as epitopes todistinguish between naïve and positive samples. Using these 5 peptidesas classifiers, the results for the “post” or infected samples for the12 animals was divided by the “pre” or non-infected sample. The log 2ratio of the fold over naïve was calculated for the 12 animals, and thesum of these 5 ratios calculated. The sum of the log 2 ratios is shownin Table 2. Importantly, animal 28647 that was negative by every methodof testing showed no difference in the “pre” and “post” measurements,while the remaining 11 samples scored greater than 6 (at least a 2-foldincrease in the “pre” to “post” measurement for several of the 5classifier peptides).

TABLE 2 TB analysis summary for NHPs from ONPRC. Twelve NHPs wereassessed for TB disease using TST, PRIMAGRAM, Lateral Flow (PrimaTBSAT-Pak), ESAT-6 ELISA, tubercule lesions, M. tb culture, and PCRanalysis for M. tb gene sequences. Results from this work are shown asIntuitive Bio. Analysis was determined by the sum of the fold-change(FC) of specific peptides compared to negative controls (see text).Analysis results: Red: positive (+), Yellow: weak positive (+/−), Green:negative (−), and No color: not analyzed (N/A).

Example 2

This example demonstrates the use of peptide microarrays to discovernovel peptide biomarkers. Briefly, JPT microarrays were incubated withdiluted serum from M. tb infected and normal NHPs and absorbed Abdetected with biotinylated anti-simian IgG. Anti-biotin IgG-goldconjugate and SilverQuant® silver deposition technology detected thebiotin on the microarray slides. Slides with silver spots were imagedusing the AthenaQuant® System, scanned, and analyzed to quantify thesilver spots. Relative spot intensities from infected animals werecompared to normal samples to identify peptides that best differentiatedinfected from normal. These peptides were synthesized, conjugated toBSA, and the discrimination verified in an Ag microarray usingadditional serum samples and the same detection technology.

Methods

Screening Peptide Microarrays—The initial Ab screen employed a selectedsegment of the M. tb proteome, determined from the sequenced genome ofthe reference strain M. tb H37Rv. The peptide microarray consisted of6,912 15-aa peptides, each with a 12-aa overlap. Based on the results,JPT constructed a custom peptide micro-array with 1,075 15-aa peptides,each with a 3-aa overlap, that encompass 246 different M. tb proteins(secondary screen). The initial screen contained 1 array/slide (i.e.,for 1 sample); the custom array had 4 sub-arrays per slide.

Verification Peptide Microarray—Based on the initial and secondaryscreening results, 20 peptides were synthesized with a carboxyl terminalcysteine and conjugated to BSA through a NHS-maleimide linker (PierceBio-technologies) and arrayed, both individually and mixed, at optimalconcentrations and printing formulations onto Protein Microarray Slides(IBI) in a 24 sub-array format. From these experiments, 4 peptide-BSAconjugates were mixed, each at 25 μg/ml in Array Print Buffer (IBI) andarrayed onto Protein Microarray Slides along with other viral Ag fromthe CSA: Simian Basic (IBI).

Serum samples—The initial Ab screen was performed in 2 separateexperiments using serum samples from rhesus macaques donated by thePathogen Detection Laboratory at the California NPRC (CNPRC; Universityof California-Davis). A total of 4 positive serum samples were analyzed;3 samples from an experimental infection time-course experiment [2, 4,and 20 weeks post-infection (WPI)] and 1 sample from a macaque thattested positive by the TST, PCR, and culture (natural infection). Thepre-infection sample was used as the negative control serum.

The secondary screen using the custom array was performed using samplesdonated by the Pathogen Detection Laboratory at CNPRC, the OregonNPRC(ONPRC; Oregon Health and Science University, Beaverton, Oreg.), andTulane NRPC (TNPRC; Tulane University). Positive serum samples from theONPRC were derived from 12 animals that were initially quarantined, theneuthanized and necropsied following an M. tb outbreak (naturalinfection) in a colony. The negative control sera from the same animalswere from stored collections. Positive and negative (pre-infection)samples from TNPRC were drawn from an experimental infection time-courseexperiment with 5-6 points from 6 animals. In addition, 56 negativesamples from the Wisconsin NPRC and the Caribbean NPRC were alsoanalyzed in the secondary screen.

Screening Microarray Assay Method—For the initial screen, the peptidemicroarray slides were assayed using the CSA: Simian Detection kit(Prod. No. 12-1006, Intuitive Biosciences) protocol. Briefly, Abs indiluted serum were permitted to bind to peptide Ags and were detectedwith a combination of biotinylated anti-simian IgG Ab and anti-biotinIgG-gold conjugate. Ab-gold complexes were visualized by addingSilverQuant® Reagents, the slides quickly washed with water and dried.The slides were imaged with the AthenaQuant System by IntuitiveBiosciences, and scanned. Silver spots in the images were identified,quantified, and the background subtracted using AthenaQuant® software(Intuitive Biosciences). Quantified spot intensities were reported asrelative intensity units (RIU) in Excel and matched to the appropriatepeptide spot using a GAL (GenePix Array List) file from JPT (except forthe verification assays).

Results

For the primary screening assay, we calculated the average RIU oftriplicate spots for each peptide for each sample then divided thisaverage by the average of the negative control sample for the samepeptide. For each peptide, we calculated the sum of the fold change overthe negative control ratio by adding the individual ratios of 2, 4, and20 WPI with the natural infection sample to a Sum Fold Change (data notshown). We selected the top 756 peptides for further evaluation andinclusion on the secondary screening arrays.

The entire collection of available serum samples was assayed using thesecondary screen peptide microarray. Heilman Consulting (Madison, Wis.)used quantile normalization and principal components analysis (PCA) toanalyze 88 positive and 68 negative samples from ONPRC, CNPRC, andTNPRC. Infected and normal samples were compared using a Two-SampleT-Test and a Mann-Whitney (MW) Rank Sum Test. The 20 peptides with thehighest rank are summarized in Table 3. Some peptides originate fromwell-known antigenic proteins, such as ESAT-6, while others are derivedfrom proteins with unknown function and previously not known forimmunogenicity in NHPs.

TABLE 3 Top 20 peptides. Using Two-Sample T-Test and a Mann-Whitney RankSum Test, peptides were assessed for discrimination of M. tb positiveserum samples compared to negative samples. Peptide Gene name ProteinName 1 Rv0104 conserved hypothetical protein 2 Rv2873 cell surfacelipoprotein MPT83 3 Rv1037c, putative ESAT-6 like protein Rv1198,Rv3619c 4 Rv2497c putative alpha-keto acid dehydrogenase 5 Rv0918conserved hypothetical protein 6 Rv3875 ESAT-6 7 Rv3286c sigF, sigmafactor F 8 Rv1509 conserved hypothetical protein 9 Rv0099 possiblefatty-acid-CoA synthetase 10 Rv0040c 28 kDa antigen 11 Rv0019 conservedhypothetical protein 12 Rv1926c MPT63 13 Rv3879c espK 14 Rv3871 ESXconserved, unknown function 15 Rv3875 ESAT-6 16 Rv0085 putativehydrogenase 17 Rv1009 putative resuscitation promoting factor 18 Rv2006putative trehalose-6-phosphate phosphatase 19 Rv0050 putative ponA1,penicillin binding protein 20 Rv3874 CFP-10

The samples acquired from the ONPRC had positive TST results duringroutine screening at the facility [personal communication, K. Andrews].After one animal presented a positive TST, it was immediatelyquarantined along with the NHPs that were housed in the same enclosure—atotal of 12 animals. Additional tests were performed in parallel on thequarantined animals. Of the 12 animals, 7 tested positive by TST(performed at the ONPRC). All 12 were then tested with a PrimaTBSTAT-Pak lateral flow device (Chembio Diagnostic Systems), an IFGA(PRIMAGAM), and an ESAT-6 ELISA. Each animal was euthanized and anecropsy performed. M. tb culture was performed at National VeterinaryServices Laboratories (Ames, Iowa) and PCR analysis at Washington AnimalDisease Diagnostic Lab (WADDL). Finally, gross pathology of lungs wasperformed during necropsy to identify lesions indicative oftuberculosis. Table 2 above shows the results of the analyses anddemonstrates the inability of current assays to conclusively identify atrue tuberculosis infection as indicated by the necropsy results.

Serum samples from the same 12 NHPs were assayed using the secondaryscreen peptide microarray. The fold change (FC) increase in signalintensity for each peptide was calculated by dividing the RIU for theTB-infected sample by the mean of the 40 negative samples (pre-infectionplus TST negatives). The sum of the FC for 20 individual peptides wascalculated for each animal and shown in Table 2 as negative (<30), weakpositive (30-45), and positive (>45). Using these qualifications, ourassay results showed 11 of 12 animals had Abs specific to these 20peptides.

Samples from experimentally TB-infected animals at TNPRC were run onIBI's verification peptide microarrays to determine each peptide'sutility at identifying positive samples. Using 141 negatives and 35positives (experimental and natural infections), a receiver operatingcharacteristic (ROC) analysis was performed for each peptide (data notshown). Peptides 4, 6, 15, and 20 provided the best sensitivity andspecificity, but no single peptide had >90% sensitivity. To improve thissensitivity, we pooled equal volumes of the 4 peptides (Mix A) to createan Ag mix that detected all positive samples (FIG. 1). For example,Sample CA75 was detected by Peptide 20 alone and Sample HI36 wasdetected by Peptide 15 alone. Notably, only 4 out of 20 peptides coulddiscriminate between positive and negative serum samples. The Mix Aformulation was optimized to reduce background signal (data not shown),arrayed, and used to screen 141 negative and 35 positive serum samples.An ROC analysis was performed and using a cutoff value of 7364 RIU, thecalculated sensitivity was 97.1% and specificity was 87.2% (FIG. 2).

Example 3

This example demonstrates that not all ESAT-6 spanning peptides areuseful to identify TB-positive animals. As can be seen in the followingtable, the first two peptides from the N-C terminus of ESAT-6 are notuseful in distinguishing between TB positive and negative groups. Thiswas taken from the second round of screening data. Peptides wereselected that had a high p-value AND Mann-Whitney rank. The peptides nothighlighted were not used any further in the development process becausethey were not statistically relevant (no change between TB and negativegroups) and had a low fold change rank. FC rank measured the magnitudeof the response.

P-value P-Value MW FC Order Probe ID (T-test) rank Rank rank 1 MTEQQWNF0.019702746 239 304 44 AGIEAAA 2 AAASAIQG 0.029676213 275 104 263NVTSIHS 3 IHSLLDEG 5.92E−06 4 36 47 KQSLTKL 4 TKLAAAWG 5.04E−06 3 1 3SGSEAY 5 EAYQGVQQ 1.04E−07 2 4 1 KWDATAT 6 ELNNALQN 3.64E−05 9 677 20LARTISE 8 QAMASTEG 0.079415726 409 687 320 NVTGMFA

Example 4

Using a mix of peptides 6, 15, 17, and 20 on the CSA: Simian arrayplatform, 6 of 12 positive samples were correctly identified. See FIG.3. Looking at signal for individual peptides, early and active infectionmay be better identified by examining the RTU for Peptides 3 and 9, inaddition to 6, 15, and 20. For latently infected animals, Peptides 2, 3,15, 19, and 20 are seroreactive and may better identify latentlyinfected animals.

REFERENCES

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The invention claimed is:
 1. An immunoassay device comprising a firstcapture agent comprising a base peptide comprising SEQ ID NO:141 and upto 5 amino acids on either or both of the amino- or carboxy-terminus ofthe base peptide, a second capture reagent comprising a base peptidecomprising SEQ ID NO:145 and up to 5 amino acids on either or both ofthe amino- or carboxy-terminus of the base peptide, a third capturereagent comprising a peptide encoding SEQ ID NO:147; and a fourthcapture reagent comprising a peptide encoding SEQ ID NO:98.
 2. Theimmunoassay device of claim 1, further comprising a surface having atleast one capture agent displayed thereon.